DESCRIPTION (the applicant's description verbatim): The rate of nucleotide turnover by the contractile protein myosin is an important determinant of the speed and power of myocardial and skeletal muscle contraction, and yet, there is little understanding of the molecular mechanisms by which the kinetic properties of myosin are regulated. Such knowledge is important (1) to explain the basis for the wide diversity of muscles in terms of contractile performance and (2) because contractile properties are significantly depressed in many disease states, for example, in end-stage congestive heart failure. We propose a series of studies designed to investigate the roles of specific regions of the myosin heavy chain (MHC) in determining the rate of nucleotide turnover in the nucleotide binding pocket. We hypothesize that a flexible loop (residues 204-216) adjacent to the pocket modulates the kinetics of nucleotide turnover, and further, that the effects of the loop on kinetics are influenced in an isoform-specific manner by the sequence of the loop and by putative electrostatic interactions of the loop with another region of the MHC (residues 323-351), the so-called interactive micro-domain (IMD). The following experiments will be done: (1) the kinetic effects of natural variations in sequence of the loop and IMD will be assessed by measuring myosin ATPase activity, rate of ADP release, and the dynamic mechanical properties of cardiac myocytes in which we will also determine the primary sequences of the loop and the IMID using single-cell PCR; (2) mechanisms underlying the variable kinetics of alpha and beta MHCs will be investigated using knock-in technology to generate mice expressing mutant MHCs in which the loop and IMD are replaced with the analogous sequences from other MHC isoforms, and then assessing the effects of these mutations on contraction, nucleotide turnover, and ADP release; (3) the mechanism by which interactions between the loop and IMD confer kinetic properties will be investigated by systematically reversing electrostatic charges in these two regions of the myosin molecule. Results from these studies should provide new insights into the mechanisms by which the work and power generating capabilities of myosin differ in various MHC isoforms.